Stabilized test device and process for detecting couplable compounds

ABSTRACT

A STABLE TEST DEVICE WHICH UNDERGOES A COLOR CHANGE WHEN CONTACTED BY A COUPLABLE COMPOUND. THE DEVICE COMPRISES A CARRIER INCORPORATING THE DRY RESIDUE OF DIAZONIUM SALT SOLUTION. THE DEVICE IS PREPARED BY CONTACTING A CARRIER WITH A DIAZONIUM SALT SOLUTION CONTAINING A STABILIZER FOLLOWED BY DRYING. THE DEVICE IS USED IN A PROCESS FOR DETECTING COUPLABLE COMPOUNDS.

United States Patent 0.

3,585,001 STABILIZED TEST DEVICE AND PROCESS FOR DETECTING COUPLABLE COMPOUNDS Raymond L. Mast, Elkhart, Ind., assignor to Miles Laboratories, Inc., Elkhart, Ind. N Drawing. Filed Feb. 17, 1969, Ser. No. 799,928 Int. Cl. G01n 21/06, 33/16 US. Cl. 23230 14 Claims ABSTRACT OF THE DISCLOSURE A stable test device which undergoes a color change when contacted by a couplable compound. The device comprises a carrier incorporating the dry residue of diazonium salt solution. The device is prepared by contacting a carrier with a diazonium salt solution containing a stabilizer followed by drying. The device is used in a process for detecting couplable compounds.

This invention relates to a stable test device which undergoes a rapid color change when contacted by a diazo couplable compound. The invention also relates to a method for preparing and using such a test device. More particularly, this invention relates to a stable test device comprising a carrier incorporating the dry residue of a diazonium salt solution and to a process for preparing said solution.

Many compounds, particularly those in body fluids such as blood or urine, are detectable by a color reaction that occurs when they are coupled with a diazo compound. One such compound which is of particular interest in the determination of a persons physical condition is bilirubin.

The detection of bilirubin in body fluids has been carried out since about 1883 when Ehrich introduced the diazo reaction for detecting bilirubin in urine. Since that time, many liquid systems employing the diazo reaction have been used for determining bilirubin in urine and serum. These systems have introduced many variations as to the diazo compounds and other reagents used in this determination. However, such systems are primarily dependent upon the azobilirubin red-violet color which forms when bilirubin reacts with a diazonium salt, which color may be measured visually or spectrophotometrically.

Although these liquid systems result in quite accurate determinations of bilirubin, they are generally ditficult to use because the diazonium salt solutions in these systems are unstable and therefore necessitate fresh preparation with each batch of fluid to be tested. Also, the time required for completion of the indicating color change is quite lengthy, the shortest being about 30 minutes and some requiring a matter of hours. Therefore, test results are not as promptly available as desired, particularly when determining bilirubin in infants.

Many of these difliculties are alleviated by a product commercially known as Ictotest, which is in the form of a dry tablet, for determination of bilirubin as described in US. Pat. No. 2,854,317. In Ictotest, the tablet comprises a diazonium salt, sulfosalicylic acid and sodium bicarbonate. To use this test, drops of urine are placed on a special highly absorbent mat. The Ictotest reagent tablet is then placed in the center of the moistened area and two drops of water flowed over the tablet. If bilirubin is present, a color reaction occurs on the mat within about 30 seconds. Although Ictotest provides a relatively rapid and stable system for determining bilirubin, a multiple number of components are required to complete the indicating reaction.

Test devices of the dip-and-read type, which combine specialized rapid reacting reagents with a suitable carrier, are widely utilized, particularly in the medical field. Such devices are commonly used for determining pH and de- "ice tecting glucose, protein and the like in body fluids. However, when the known test systems for detecting bilirubin were combined with a suitable carrier to form a dip-andread type device, the resulting device was unstable and yielded unreliable results.

Therefore, it is an object of this invention to provide a stable test device which undergoes a rapid color change when contacted by a diazo couplable compound.

It is a further object of this invention to provide a stable test device which is suitable for the detection of bilirubin in a solution.

Another object of this invention is to provide a method for preparing a unitary test device that may be used in the detection of bilirubin in a solution.

Additional objects of this invention will become evident from the following description.

This invention is embodied in a novel stable test device which undergoes a color change when contacted by a diazo couplable compound. This device comprises a carrier impregnated with a dry residue of a diazonium salt solution comprising a reaction product formed by combining an organic polar solvent, a couplable arylamine, water, a soluble nitrite, a stabilizer and a sulfonic acid.

This invention is also embodied in a process for preparing the test device, comprising contacting a preferably absorbent carrier with a diazonium salt solution and drying the carrier.

In preparing the diazonium salt solution, a polar solvent is used. Preferably, the composition is made up of water and an organic polar solvent such as an alcohol, a ketone, an ester and the like. Advantageously, an alcohol such as methanol, ethanol and 2-propanol is used in a ratio of at least about four parts organic solvent to one part water. The ratio of organic solvent to water is selected so that there is substantially no precipitate in the diazonium salt solution formed. Also, a high ratio of a rapidly evaporating organic solvent is desirable to reduce drying time of a carrier contacted with the solution.

A couplable arylamine that may be diazotized is beneficially added to the solvent first. Such an arylamine is advantageously selected from 2,4-dichloroaniline; p-nitroaniline; p-chloroaniline; 2,5-dichloroaniline; 4-chloro-oanisidine; 3,3'-dimethoxybenzidine; and 2-methoxy-5-nitroaniline. Other such arylamines that are capable of forming diazonium compounds may also be used and are considered within the scope of this invention.

A suitable soluble nitrite that will form nitrous acid in an aqueous acid medium is then added to the solution. Beneficially, the soluble nitrite is sodium nitrite, potassium nitrite or calcium nitrite.

The solution also includes a stabilizer compound. Such stabilizer has been found beneficial in that any precipitate that appears upon the formation of the diazonium salt forms as a fine precipitate which is readily dissolved. Additionally, the stabilizer has been found to add chemical stability to the final test device. The stabilizer may be selected from a broad group of compounds, such as, aromatics or aliphatic sulfonic acid salts, sulfates or sulfonates. Examples of such stabilizers include 1,5-naphthalenedisulfonic acid, disodium salt; Z-naphthalenesulfonic acid, sodium salt; 4,4'-diamino 2,2 biphenyldisulfonic acid, disodium salt and sodium lauryl sulfate.

An organic acid, preferably an organic sulfonic acid, is included in the composition to provide the proper pH for the formation of a diazonium compound and the subsequent coupling reaction of this diazonium compound with the couplable compound. The organic sulfonic acid utilized beneficially may be selected from sulfosalicylic acid, sulfamic acid, hexamic and p-toluenesulfonic acid and the like. It is understood that other known sulfonic acids and organic acids may be used within the scope of this invention.

These reagents, a polar solvent, an arylamine, water, soluble nitrite, a stabilizer and a sulfonic acid are combined to form the desired diazonium salt solution. Preferably, these reagents are combined in the order in which they have been mentioned, although the order of combination of the polar solvent, arylamine, water, soluble nitrite and stabilizer is not considered critical. Also, if more than one stabilizer is used, the second stabilizer may be added after the sulfonic acid.

Diazo dyes have been prepared using a reagent system similar to that described above. The diazo dyes prepared in the prior art systems formed as large particle precipitates which were desirable for their non-dusting properties. Such compounds Were formed at reduced temperatures, about C. Surprisingly, it has now been found that with the method of this invention a diazonium salt solution may be formed at room temperature (about 23 C.) and retained in a solution which is substantially free of precipitate at such temperature with reagents similar to those of prior art systems. Furthermore, it has been discovered that when a carrier is contacted with this diazonium salt solution and such carrier is then dried, the residue of the diazonium salt solution forms in small particles that are readily retained by the carrier.

The proportions of the reagents used to form the diazonium salt solution are preferably within the following ranges which are expressed in weight/ volume percent, as appropriate:

Percent Polar solvent 10-90 Arylamine 0.02-0.5 Water l090 Soluble nitrite 0.01-0.25 Stabilizer 0.25 Sulfonic acid 0.2-5

As previously mentioned, advantageously the ratio of solvent to water is maintained at 4:1 or higher to retain the diazonium salt in solution and promote the drying of the carrier. Also, the weight ratio of acid to arylamine compound is preferably maintained at about :1.

A suitable carrier may be utilized that is capable of retaining the dry residue of the diazonium salt solution incorporated with the carrier. This carrier is preferably mounted in such a manner that it may be readily contacted by a fiuid suspected of including the couplable compound to be detected. The carrier for the test device may be an integral part of a larger portion serving as a handle or may be a separate part that is affixed to a larger handle or handling device. Such a carrier may l comprise an absorbent material that is dipped in or otherwise contacted with the diazonium salt solution and subsequently dried. Suitable carriers include, for example, a porous material such as filter paper, glass fiber paper,

or a polypropylene felt. The carrier may also comprise I a polymeric gel which has been premixed with the test solution and dried so as to form a semipermeable membrane structure. The gel may be formed by solvent evaporation and may include a cross-linking agent.

The overall structure of the test device is not considered critical so long as a suitable means is provided for contacting the area including the dry residue of the diazonium salt solution with the fluid to be tested.

A test device may also be prepared by impregnating a piece of filter paper, which functions as a carrier, with the diazonium salt solution and drying the same. The impregnated paper is preferably dried at a moderate temperature of about 65 to 70 C. in moving air. Advantageously, the temperature is not elevated excessively as this is believed to have a deleterious effect upon the diazonium compounds. The dried carrier may then be divided into small portions and affixed to a larger handle for easier handling.

The invention will be further described in the following examples which are intended to be illustrative and are 4 not to be construed as limitations on the scope of the invention.

EXAMPLE 1 A solution was prepared by combining in a glass vessel, in the order listed and with continuous mixing, the following compounds:

The compounds when combined were at ambient room temperature (about 23 C.) and the temperature of the solution remained at about ambient room temperature without additional temperature control throughout the formation of the solution.

EXAMPLE 2 A solution was prepared as described in Example 1 except that 0.05 g. of 2,4-dichloroaniline and 0.5 g. of Sulfosalicylic acid were used. This solution was observed to have substantially the same properties as the solution of Example 1.

EXAMPLE 3 A solution was prepared as described in Example 1 except that 0.5 g. of 2,4-dichloroaniline and 5.0 g. of sulfosalicylic acid were used. This solution was observed to have substantially the same properties as the solution of Example 1.

EXAMPLES 4-10 Separate solutions were prepared as described in Example 1 except that one of the following compounds were substituted for the 2,4-dichlor0aniline.

p-nitroaniline p-chloroaniline 2,5 -dichloroaniline 4-chloro-o-anisidine 3,3 '-dimethoxybenzidine 2-methoxy-5-nitroaniline The properties of the resulting solutions were observed to be substantially the same as for the solution of Example 1.

EXAMPLE 11 A solution was prepared as described in Example 1 except that hexamic acid was used in place of Sulfosalicylic acid. The resulting solution was observed to have substantially the same properties as the solution of Example 1.

EXAMPLE 12 A solution was prepared as described in Example 1 except that sulfamic acid was used in place of sulfosalicylic acid. The resulting solution was observed to have substantially the same properties as the solution of Example 1.

EXAMPLE 13 A solution was prepared as described in Example 1 except that p-toluenesulfonic acid was used in place of sulfosalicylic acid. The resulting solution was observed to have substantially the same properties as the solution of Example 1.

EXAMPLE 14 A strip of filter paper (Eaton and Dikeman No. 641) was immersed in the solution of Example 1 and immediately removed. This paper was dried between about and C. for between about 8 and 10 minutes with air movement over the surface of the paper. The dried paper had a cream white color.

Bilirubin test solutions were prepared from a pathological urine sample obtained from a patient showing signs of jaundice. The urine obtained from the patient was treated with Ictotest and a very large reaction was observed which indicated a high concentration of bilirubin. This urine was also assayed by a modification of the E. G. Godfried procedure for bilirubin described in Biochem. J 28, 20562060, 1934 and found to have approximately 1 mg. percent bilirubin. The pathological urine was further diluted With normal urine to concentrations of 0.5, 0.25 and 0.1 mg. percent. Each solution was tested with test devices of this invention as prepared above and gradations of pink were observed. A deeper pink, approaching a pink-lavender, was observed for the solutions of higher concentrations.

Similarly, human blood sera were obtained from normal patients and patients showing jaundice. The bilirubin content of these sera, as determined with an Auto Analyzer, was between 0.1 and 18 mg. percent. Test devices prepared above were contacted with the sera and colors varying from pink to lavender were observed for concentrations of 1.0 mg. percent and higher. The color change was proportional to the concentration of bilirubin in the solutions.

EXAMPLE 15 Strips of glass fiber paper (Gelman Type A-1306 and Type E-748) were processed in place of filter paper according to the procedure of Example 14. The resulting dried glass fiber paper had an appearance substantially the same as the paper of Example 14. When contacted by the test solutions of Example 14 color changes as noted in Example 14 were observed.

EXAMPLE 16 In this example the procedure of Example 14 was followed except that a polypropylene felt (Polypropylene MDSE. No. Po-8110 by American Felt Company), was used in place of filter paper. The dried matrix had substantially the same color appearance and color reaction to the test solutions as observed in Example 14.

EXAMPLE 17 A solution was prepared according to the proportions of Example 1. A 4-inch wide sheet of Eaton and Dikeman No. 641 filter paper was passed through this solution and dried between about 65 and 70 C. for about 10 minutes with circulating air. After drying, the paper was contacted with one pressure sensitive adhesive surface of a double-faced pressure sensitive cellulosic fiber reinforced adhesive tape, slit longitudinally into strips each inch wide and wound into rolls. A roll was then mounted on an axis and a wax paper backing or liner was removed from the outer adhesive surface of the double-faced pressure sensitive tape bonded to the paper. The exposed surface of the pressure sensitive tape was continuously bonded for the length of the roll to a surface of a 3% inch wide sheet of polystyrene film 0.01 inch thick. The pressure sensitive tape was securely bonded to the film. The laminate Was cut across the length of the strip into strips /5 inch long. The device so formed was contacted with the test solutions of Example 14 and a color change was noted as in Example 14.

What is claimed is:

1. A stable test device which exhibits a color change when contacted by a diazo couplable compound, said device comprising a carrier incorporating a dry residue of a stable diazonium salt solution comprising a reaction product formed by combining an organic polar solvent, an arylamine capable of forming a diazonium salt, water, a soluble nitrite, a sulfonic acid and a stabilizer which is selected from aromatic or aliphatic sulfonic acid salts, sulfates or sulfonates.

2. A stable test device according to claim 1 in which the carrier is an absorbent material.

3. A stable test device according to claim 1 in which the polar solvent is an alcohol, an ether, a ketone or an ester.

4. A stable test device according to claim 1 in which the arylamine is 2,4-dichloroaniline, p-nitroaniline, pchloroaniline, 2,5-dichloroaniline, 4-chloro-o-anisidine, 3,3-dimethyoxybenzidine, or 2-methoxy-5-nitroaniline.

5. A stable test device according to claim 1 in which the soluble nitrite is sodium nitrite, potassium nitrite or calcium nitrite.

6. A stable test device according to claim 1 in which the aromatic sulfonic acid salt is a member selected from the group consisting of 1,5-napthalenedisulfonic acid, disodium salt; 2-napthalenesulfonic acid, sodium salt; and 4,4-diamino-2,2'-biphenyldisulfonic acid, disodium salt.

7. A stable test device according to claim 1 in which the stabilizer is sodium lauryl sulfate.

8. A stable test device according to claim 1 in which the sulfonic acid is a member selected from the group consisting of sulfosalicylic acid, sulfamic acid, and hexamic acid.

9. A stable test device according to claim 1 in which the polar solvent is methanol; the arylamine is 2,4-dichloroaniline; the soluble nitrite is sodium nitrite, the stabilizers are 1,5-napthalenedisulfonic acid, disodium salt and sodium lauryl sulfate and the sulfonic acid is sulfosalicylic acid.

10. A stable test device according to claim 1 in which the carrier is an absorbent material; the polar solvent is an alcohol, ether, ketone or ester; the arylamine is 2,4-dichloroaniline, 4,4'-methylenebis (N,N-dimethylaniline), pnitroaniline, p-chloroaniline, 2,5-dichloroaniline, 4-chloroo-anisidine, 3,3'-dimethoxybenzidine, or 2-methoxy-5- nitroaniline; the soluble nitrite is sodium nitrite; the stabilizer is 1,5-napthalenedisulfonic acid, disodium salt; Z-napthalene sulfonic acid, sodium salt, 4,4'diamino-2,2'- biphenyldisulfonic acid, disodium salt or sodium lauryl sulfate and the sulfonic acid is sulfosalicylic acid, sulfamic acid or hexamic acid.

11. A process for preparing the test device of claim 1 comprising mixing in the order stated the polar solvent, the arylamine, the soluble nitrite, the stabilizer and the sulfonic acid to form the stable diazonium salt solution, contacting a carrier with the diazonium salt solution and drying the carrier leaving a dry residue of the diazonium salt solution thereon.

12. A stable test device according to claim 1 in which the arylamine is selected from aniline, substituted aniline, diaminobiphenyl or substituted diaminobiphenyl.

13. A process for detecting a diazo couplable compound in a solution comprising contacting the test device of claim 1 with the solution and observing the color change of the test device.

14. A process according to claim 13 in which the diazo couplable compound to be detected is bilirubin in an aqueous solution.

References Cited UNITED STATES PATENTS 2,854,317 9/1958 Free 23-253 3,375,079 3/1968 Lyshkow 252-408 3,388,075 6/1968 Braver 23-253 3,446,599 5/1969 Shand 23-253 3,477,818 11/1969 Fried 23230 JOSEPH SCOVRONEK, Primary Examiner S. MARANTZ, Assistant Examiner US. Cl. X.R. 23-253; 252-408 

